Case report: tularaemia in a white-handed gibbon (Hylobates lar), Germany.

In 2021, a white-handed gibbon (Hylobates lar) succumbed to illness shortly after transfer from one zoo to another in Germany, due to Francisella tularensis subsp. holarctica infection. To determine the source of infection, whole genome sequencing of the gibbon-derived isolate was performed and wild pest rodents (and captive squirrels) from both zoos were screened for F. tularensis. The F. tularensis whole genome sequence obtained from the gibbon was closely related to previous subclade B.281 sequences obtained from hares from Baden-Wuerttemberg, the same region where the gibbon was first housed. However, F. tularensis DNA was detected in one Norway rat from the receiving zoo. Therefore, neither zoo can be excluded as the source of infection.

Whole genome sequence-based analysis of Staphylococcus aureus isolated from bovine mastitis in Thuringia, Germany.

Background: Bovine mastitis is a common disease of dairy cattle causing major economic losses due to reduced yield and poor quality of milk worldwide. The current investigation aimed to gain insight into the genetic diversity, antimicrobial resistance profiles and virulence associated factors of Staphylococcus (S.) aureus isolated from clinical bovine mastitis in dairy farms in Thuringia, Germany. Methods: Forty Staphylococcus aureus isolates collected from clinical bovine mastitis cases from 17 Thuringian dairy farms were phenotyped and genetically characterized using whole genome sequencing. Results: Out of 40 S. aureus, 30 (75%) were confirmed as methicillin resistant isolates. The isolates showed elevated antimicrobial resistance against penicillin, tetracycline and oxacillin, i.e., 77.5, 77.5, and 75%, respectively. Lower resistance rates were found against moxifloxacin, ciprofloxacin, gentamicin and trimethoprim/sulfamethoxazole, i.e., 35, 35, 30, and 22.5%, respectively. While resistance against clindamycin and erythromycin was rarely found (5 and 2.5%, respectively). All isolates were susceptible to linezolid, teicoplanin, vancomycin, tigecycline, fosfomycin, fusidic acid and rifampicin. These isolates were further allocated into five different sequence types: ST398 (n = 31), ST1074 (n = 4), ST504 (n = 3), ST582 (CC15) (n = 1) and ST479 (n = 1). These isolates were also assigned to seven clusters with up to 100 SNP which has facilitated geographical mapping and epidemiological distribution in Thuringia. Strains belonging to ST398 were classified into clusters 1, 2, 3, 4 and 7. The isolates of ST504 were of cluster 5, those of ST1074 were belonging to cluster 6. Resistance genes blaZ, blaI and blaR associated with penicillin resistance were found in 32 (80%) strains, all except one were belonging to ST398. Methicillin resistance associated mecA was identified in 30 (96.8%) isolates of ST398. All tetracycline and erythromycin resistant isolates were of ST398, and all harbored both tetM and ermA. About 90.3% of tetracycline resistant isolates assigned to ST398 were also carrying tetK gene. The point mutations parC_S80F, gyrA_S84L and parC_S80Y in gyrA and parC associated with quinolone resistance were found in all phenotypically resistant isolates to ciprofloxacin and moxifloxacin (n = 14). Sixty-eight virulence genes were identified among isolates. Both lukD/E and lukM/F-PV-P83 were identified in 22.5% of isolates, all were non-ST398. Conclusion: In this study, ST398 had the highest potential to cause disease and had a massive prevalence in bovine mastitis cases. Five different sequence types and seven clusters were identified in the federal state of Thuringia. The circulation of some clusters in the same region over several years shows the persistence of cluster-associated infection despite the intensive veterinary care. On the other hand, some regions had different clusters at the same year or in different consecutive years. Different sequence types and associated different clusters of S. aureus were geographically widely distributed among dairy farms in Thuringia. The findings of this study show that various clusters have the potential to spread over a large geographical scale. The detection of LA-MRSA on dairy farms, which is known for cabapility to widely spread among different groups of animals, humans and their environment urges for the implementation of national wide strategic programs. The identification of CA-MRSA among the isolates such as ST398 poses a significant risk for the transmission of such strains between animals and humans on dairy farms.

Clonal relation between Salmonella enterica subspecies enterica serovar Dublin strains of bovine and food origin in Germany.

Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) is a host-adapted serovar causing enteritis and/or systemic diseases in cattle. As the serovar is not host-restricted, it may cause infections in other animals, including humans with severe illness and higher mortality rates than other non-typhoidal serovars. As human infections are mainly caused by contaminated milk, milk products and beef, information on the genetic relationship of S. Dublin strains from cattle and food should be evaluated. Whole-genome sequencing (WGS) of 144 S. Dublin strains from cattle and 30 strains from food origin was performed. Multilocus sequence typing (MLST) revealed mostly sequence type ST-10 from both, cattle and food isolates. In total, 14 of 30 strains from food origin were clonally related to at least one strain from cattle, as detected by core-genome single nucleotide polymorphisms typing as well as core-genome MLST. The remaining 16 foodborne strains fit into the genome structure of S. Dublin in Germany without outliers. WGS proved to be a powerful tool not only to gain information on the epidemiology of Salmonella strains but also to detect clonal relations between organisms isolated from different stages of production. This study has shown a high genetic correlation between S. Dublin strains from cattle and food and, therefore, the potential to cause human infections. S. Dublin strains of both origins share an almost identical set of virulence factors, emphasizing their potential to cause severe clinical manifestations in animals, but also in humans and thus the need for effective control of S. Dublin in a farm-to-fork strategy.

Oxford nanopore technologies-a valuable tool to generate whole-genome sequencing data for in silico serotyping and the detection of genetic markers in Salmonella.

Bacteria of the genus Salmonella pose a major risk to livestock, the food economy, and public health. Salmonella infections are one of the leading causes of food poisoning. The identification of serovars of Salmonella achieved by their diverse surface antigens is essential to gain information on their epidemiological context. Traditionally, slide agglutination has been used for serotyping. In recent years, whole-genome sequencing (WGS) followed by in silico serotyping has been established as an alternative method for serotyping and the detection of genetic markers for Salmonella. Until now, WGS data generated with Illumina sequencing are used to validate in silico serotyping methods. Oxford Nanopore Technologies (ONT) opens the possibility to sequence ultra-long reads and has frequently been used for bacterial sequencing. In this study, ONT sequencing data of 28 Salmonella strains of different serovars with epidemiological relevance in humans, food, and animals were taken to investigate the performance of the in silico serotyping tools SISTR and SeqSero2 compared to traditional slide agglutination tests. Moreover, the detection of genetic markers for resistance against antimicrobial agents, virulence, and plasmids was studied by comparing WGS data based on ONT with WGS data based on Illumina. Based on the ONT data from flow cell version R9.4.1, in silico serotyping achieved an accuracy of 96.4 and 92% for the tools SISTR and SeqSero2, respectively. Highly similar sets of genetic markers comparing both sequencing technologies were identified. Taking the ongoing improvement of basecalling and flow cells into account, ONT data can be used for Salmonella in silico serotyping and genetic marker detection.

Whole genome sequencing of Salmonella enterica serovars isolated from humans, animals, and the environment in Lagos, Nigeria.

BACKGROUND: Salmonella infections remain an important public health issue worldwide. Some serovars of non-typhoidal Salmonella (NTS) have been associated with bloodstream infections and gastroenteritis, especially in children in Sub-Saharan Africa with circulating S. enterica serovars with drug resistance and virulence genes. This study identified and verified the clonal relationship of Nigerian NTS strains isolated from humans, animals, and the environment. METHODS: In total, 2,522 samples were collected from patients, animals (cattle and poultry), and environmental sources between December 2017 and May 2019. The samples were subjected to a standard microbiological investigation. All the isolates were identified using Microbact 24E, and MALDI-TOF MS. The isolates were serotyped using the Kauffmann-White scheme. Antibiotic susceptibility testing was conducted using the disc diffusion method and the Vitek 2 compact system. Virulence and antimicrobial resistance genes, sequence type, and cluster analysis were investigated using WGS data. RESULTS: Forty-eight (48) NTS isolates (1.9%) were obtained. The prevalence of NTS from clinical sources was 0.9%, while 4% was recorded for animal sources. The serovars identified were S. Cotham (n = 17), S. Give (n = 16), S. Mokola (n = 6), S. Abony (n = 4), S. Typhimurium (n = 4), and S. Senftenberg (n = 1). All 48 Salmonella isolates carried intrinsic and acquired resistant genes such as aac.6…Iaa, mdf(A), qnrB, qnrB19 genes and golT, golS, pcoA, and silP, mediated by plasmid Col440I_1, incFIB.B and incFII. Between 100 and 118 virulence gene markers distributed across several Salmonella pathogenicity islands (SPIs), clusters, prophages, and plasmid operons were found in each isolate. WGS revealed that strains of each Salmonella serovar could be assigned to a single 7-gene MLST cluster, and strains within the clusters were identical strains and closely related as defined by the 0 and 10 cgSNPs and likely shared a common ancestor. The dominant sequence types were S. Give ST516 and S. Cotham ST617. CONCLUSION: We found identical Salmonella sequence types in human, animal, and environmental samples in the same locality, which demonstrates the great potential of the applied tools to trace back outbreak strains. Strategies to control and prevent the spread of NTS in the context of one's health are essential to prevent possible outbreaks.

Comparison of Illumina and Oxford Nanopore Technology for genome analysis of Francisella tularensis, Bacillus anthracis, and Brucella suis.

BACKGROUND: Bacterial epidemiology needs to understand the spread and dissemination of strains in a One Health context. This is important for highly pathogenic bacteria such as Bacillus anthracis, Brucella species, and Francisella tularensis. Whole genome sequencing (WGS) has paved the way for genetic marker detection and high-resolution genotyping. While such tasks are established for Illumina short-read sequencing, Oxford Nanopore Technology (ONT) long-read sequencing has yet to be evaluated for such highly pathogenic bacteria with little genomic variations between strains. In this study, three independent sequencing runs were performed using Illumina, ONT flow cell version 9.4.1, and 10.4 for six strains of each of Ba. anthracis, Br. suis and F. tularensis. Data from ONT sequencing alone, Illumina sequencing alone and two hybrid assembly approaches were compared. RESULTS: As previously shown, ONT produces ultra-long reads, while Illumina produces short reads with higher sequencing accuracy. Flow cell version 10.4 improved sequencing accuracy over version 9.4.1. The correct (sub-)species were inferred from all tested technologies, individually. Moreover, the sets of genetic markers for virulence, were almost identical for the respective species. The long reads of ONT allowed to assemble not only chromosomes of all species to near closure, but also virulence plasmids of Ba. anthracis. Assemblies based on nanopore data alone, Illumina data alone, and both hybrid assemblies correctly detected canonical (sub-)clades for Ba. anthracis and F. tularensis as well as multilocus sequence types for Br. suis. For F. tularensis, high-resolution genotyping using core-genome MLST (cgMLST) and core-genome Single-Nucleotide-Polymorphism (cgSNP) typing produced highly comparable results between data from Illumina and both ONT flow cell versions. For Ba. anthracis, only data from flow cell version 10.4 produced similar results to Illumina for both high-resolution typing methods. However, for Br. suis, high-resolution genotyping yielded larger differences comparing Illumina data to data from both ONT flow cell versions. CONCLUSIONS: In summary, combining data from ONT and Illumina for high-resolution genotyping might be feasible for F. tularensis and Ba. anthracis, but not yet for Br. suis. The ongoing improvement of nanopore technology and subsequent data analysis may facilitate high-resolution genotyping for all bacteria with highly stable genomes in future.

Genomic insight into Campylobacter jejuni isolated from commercial turkey flocks in Germany using whole-genome sequencing analysis.

Campylobacter (C.) jejuni is a zoonotic bacterium of public health significance. The present investigation was designed to assess the epidemiology and genetic heterogeneity of C. jejuni recovered from commercial turkey farms in Germany using whole-genome sequencing. The Illumina MiSeq® technology was used to sequence 66 C. jejuni isolates obtained between 2010 and 2011 from commercial meat turkey flocks located in ten German federal states. Phenotypic antimicrobial resistance was determined. Phylogeny, resistome, plasmidome and virulome profiles were analyzed using whole-genome sequencing data. Genetic resistance markers were identified with bioinformatics tools (AMRFinder, ResFinder, NCBI and ABRicate) and compared with the phenotypic antimicrobial resistance. The isolates were assigned to 28 different sequence types and 11 clonal complexes. The average pairwise single nucleotide-polymorphisms distance of 14,585 SNPs (range: 0-26,540 SNPs) revealed a high genetic distinction between the isolates. Thirteen virulence-associated genes were identified in C. jejuni isolates. Most of the isolates harbored the genes flaA (83.3%) and flaB (78.8%). The wlaN gene associated with the Guillain-Barré syndrome was detected in nine (13.6%) isolates. The genes for resistance to ampicillin (bla OXA), tetracycline [tet(O)], neomycin [aph(3')-IIIa], streptomycin (aadE) and streptothricin (sat4) were detected in isolated C. jejuni using WGS. A gene cluster comprising the genes sat4, aph(3')-IIIa and aadE was present in six isolates. The single point mutation T86I in the housekeeping gene gyrA conferring resistance to quinolones was retrieved in 93.6% of phenotypically fluoroquinolone-resistant isolates. Five phenotypically erythromycin-susceptible isolates carried the mutation A103V in the gene for the ribosomal protein L22 inferring macrolide resistance. An assortment of 13 ß-lactam resistance genes (bla OXA variants) was detected in 58 C. jejuni isolates. Out of 66 sequenced isolates, 28 (42.4%) carried plasmid-borne contigs. Six isolates harbored a pTet-like plasmid-borne contig which carries the tet(O) gene. This study emphasized the potential of whole-genome sequencing to ameliorate the routine surveillance of C. jejuni. Whole-genome sequencing can predict antimicrobial resistance with a high degree of accuracy. However, resistance gene databases need curation and updates to revoke inaccuracy when using WGS-based analysis pipelines for AMR detection.

Epidemiological Analysis on the Occurrence of Salmonella enterica Subspecies enterica Serovar Dublin in the German Federal State Schleswig-Holstein Using Whole-Genome Sequencing.

The cattle-adapted serovar Salmonella Dublin (S. Dublin) causes enteritis and systemic diseases in animals. In the German federal state Schleswig-Holstein, S. Dublin is the most important serovar in cattle indicating an endemic character of the infection. To gain information on dissemination and routes of infection, whole-genome sequencing (WGS) was used to explore the genetic traits of 78 S. Dublin strains collected over a period of six years. The phylogeny was analysed using core-genome single nucleotide polymorphisms (cgSNPs). Genomic clusters at 100, 15 and 1 cgSNPs were selected for molecular analysis. Important specific virulence determinants were detected in all strains but multidrug resistance in S. Dublin organisms was not found. Using 15 cgSNPs epidemiological links between herds were identified, clusters at 1 cgSNPs provided clear evidence on both persistence of S. Dublin at single farms in consecutive years and transmission of the organisms between herds in different distances. A possible risk factor for the repeated occurrence of S. Dublin in certain districts of Schleswig-Holstein might be the spreading of manure on pastures and grassland. Effective control of S. Dublin requires farm-specific analysis of the management supplemented by WGS of outbreak causing S. Dublin strains to clearly identify routes of infection.

[Assessment of available and currently applied typing methods including genome-based methods for zoonotic pathogens with a focus on Salmonella enterica]. / Bestandsaufnahme der verfügbaren und aktuell eingesetzten Typisierungsmethoden einschließlich genombasierter Verfahren von Zoonoseerregern am Beispiel von Salmonella enterica.

BACKGROUND: In recent years, whole genome sequencing (WGS) in combination with bioinformatic analyses has become state of the art in evaluating the pathogenicity/resistance potential and relatedness of bacteria. WGS analysis thus represents a central tool in the investigation of the resistance and virulence potential of pathogens, as well as their dissemination via outbreak clusters and transmission chains within the framework of molecular epidemiology. In order to gain an overview of the available genotypic and phenotypic methods used for pathogen typing of Salmonella and Shiga toxin-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany at state and federal level, along with the availability of WGS-based typing and corresponding analytical methods, a survey of laboratories was conducted. METHODS: An electronic survey of laboratories working for public health protection and consumer health protection was conducted from February to June 2020. RESULTS AND CONCLUSION: The results of the survey showed that many of the participating laboratories provide a wide range of phenotypic and molecular methods. Molecular typing is most commonly used for species identification of Salmonella. In many cases, WGS-based methods have already been established at federal and state institutions or are in the process of being established. The Illumina sequencing technology is the most widely used technology. The survey confirms the importance of molecular biology and whole genome typing technologies for laboratories in the diagnosis of bacterial zoonotic pathogens.

Tracking the distribution, genetic diversity and lineage of Brucella melitensis recovered from humans and animals in Egypt based on core-genome SNP analysis and in silico MLVA-16.

Brucellosis is one of the most common neglected zoonotic diseases globally, with a public health significance and a high economic loss in the livestock industry caused by the bacteria of the genus Brucella. In this study, 136 Egyptian Brucella melitensis strains isolated from animals and humans between 2001 and 2020 were analysed by examining the whole-core-genome single-nucleotide polymorphism (cgSNP) in comparison to the in silico multilocus variable number of tandem repeat analysis (MLVA-16). Almost all Egyptian isolates were belonging to the West Mediterranean clade, except two isolates from buffalo and camel were belonging to the American and East Mediterranean clades, respectively. A significant correlation between the human case of brucellosis and the possible source of infection from animals was found. It seems that several outbreak strains already existing for many years have been spread over long distances and between many governorates. The cgSNP analysis, in combination with epidemiological metadata, allows a better differentiation than the MLVA-16 genotyping method and, hence, the source definition and tracking of outbreak strains. The MLVA based on the currently used 16 markers is not suitable for this task. Our results revealed 99 different cgSNP genotypes with many different outbreak strains, both older and widely distributed ones and rather newly introduced ones as well. This indicates several different incidents and sources of infections, probably by imported animals from other countries to Egypt. Comparing our panel of isolates to public databases by cgSNP analysis, the results revealed near relatives from Italy. Moreover, near relatives from the United States, France, Austria and India were found by in silico MLVA.

Sequence Analysis of Novel Staphylococcus aureus Lineages from Wild and Captive Macaques.

Staphylococcus aureus is a widespread and common opportunistic bacterium that can colonise or infect humans as well as a wide range of animals. There are a few studies of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from monkeys, apes, and lemurs, indicating a presence of a number of poorly or unknown lineages of the pathogen. In order to obtain insight into staphylococcal diversity, we sequenced strains from wild and captive individuals of three macaque species (Macaca mulatta, M. assamensis, and M. sylvanus) using Nanopore and Illumina technologies. These strains were previously identified by microarray as poorly or unknown strains. Isolates of novel lineages ST4168, ST7687, ST7688, ST7689, ST7690, ST7691, ST7692, ST7693, ST7694, ST7695, ST7745, ST7746, ST7747, ST7748, ST7749, ST7750, ST7751, ST7752, ST7753, and ST7754 were sequenced and characterised for the first time. In addition, isolates belonging to ST2990, a lineage also observed in humans, and ST3268, a MRSA strain already known from macaques, were also included into the study. Mobile genetic elements, genomic islands, and carriage of prophages were analysed. There was no evidence for novel host-specific virulence factors. However, a conspicuously high rate of carriage of a pathogenicity island harbouring edinB and etD2/etE as well as a higher number of repeat units within the gene sasG (encoding an adhesion factor) than in human isolates were observed. None of the strains harboured the genes encoding Panton-Valentine leukocidin. In conclusion, wildlife including macaques may harbour an unappreciated diversity of S. aureus lineages that may be of clinical relevance for humans, livestock, or for wildlife conservation, given the declining state of many wildlife populations.

Sequencing-Based Genotyping of Pakistani Burkholderia mallei Strains: A Useful Way for Investigating Glanders Outbreaks.

Burkholderia (B.) mallei is a host-adapted equine pathogen that causes glanders, a re-emerging zoonotic disease, which is endemic in Pakistan and other developing countries and seriously impacts the global equine movement. Due to globalization, the geographical restriction of diseases vanishes and the lack of awareness of and experience with eradicated diseases in industrialized countries also promotes the re-introduction of infections in these regions. Owing to the high equine population, the Pakistani province Punjab is a potential hotspot where several glanders outbreaks have been seen over last two decades. For determining the genomic diversity of B. mallei in this and other equine-populated prefectures, the genomes of 19 B. mallei strains isolated between 1999 and 2020 in different locations were sequenced and their genotypes were determined. Particularly, for genetically highly homogenous pathogens like B. mallei genotyping techniques require a high discriminatory power for enabling differentiation on the strain level. Thus, core-genome single nucleotide polymorphism (cgSNP) analysis was applied for distinguishing the highly similar strains. Furthermore, a whole-genome sequence-based core genome multi locus sequence typing (cgMLST) scheme, specific to B. mallei, was developed and additionally applied to the data. It was found that B. mallei genotypes in Pakistan persisted over time and space and genotype clusters preferred connection with a time point rather than the place of isolation, probably due to frequent equine movement, which promotes the spread of glanders. The cgMLST approach proved to work in accord with SNP typing and may help to investigate future glanders outbreaks.

WGS-Based Phenotyping and Molecular Characterization of the Resistome, Virulome and Plasmid Replicons in Klebsiella pneumoniae Isolates from Powdered Milk Produced in Germany.

The emergence of Klebsiella pneumoniae (K. pneumoniae) in German healthcare is worrying. It is not well-investigated in the veterinary world and food chains. In the current study, antibiotic susceptibility profiles of 24 K. pneumoniae strains isolated from powdered milk samples produced in Germany were investigated by a microdilution test. Next-generation sequencing (NGS) was applied to identify genomic determinants for antimicrobial resistance (AMR), virulence-associated genes and plasmids replicons. All isolates were susceptible to the majority (14/18) of tested antibiotics. Resistance to colistin, fosfomycin, chloramphenicol and piperacillin was found. The ambler class A ß-lactamase, blaSHV variants were identified in all isolates, of which blaSHV-187 was most prevalent and found in 50% of isolates. Single-nucleotide-variants of oqxA and oqxB conferring resistance to phenicol/quinolone were found in all isolates, and the oqxB17 was the most prevalent found in 46% of isolates. 67% of isolates harbored fosA genes; however, only one was fosfomycin-resistant. Two isolates harbored genes conferring resistance to colistin, despite being susceptible. The majority of identified virulome genes were iron uptake siderophores. Two enterobactins (entB, fepC), six adherence-related genes belonging to E. coli common pilus (ECP) and one secretion system (ompA gene) were found in all isolates. In contrast, yersiniabactin was found in two isolates. One ST23 strain was susceptible to all tested antibiotics, and harbored determinants discriminatory for hypervirulent strains, e.g., aerobactin, salmochelin, yersiniabactin, enterobactin and regulator of mucoid phenotype A genes that are highly associated with hypervirulent K. pneumoniae. The IncF plasmid family was found in all strains, while almost half of the isolates harbored Col440I-type plasmids and nine isolates harbored various Inc-type plasmids. The presence of K. pneumoniae carrying different resistomes and major virulent specific virulomes in powdered milk samples is alarming. This could threaten public health, particularly of neonates and infants consuming dried milk.

Molecular Typing of Ukrainian Bacillus anthracis Strains by Combining Whole-Genome Sequencing Techniques.

Anthrax is a recurrent zoonosis in the Ukraine with outbreaks occurring repeatedly in certain areas. For determining whether several Bacillus anthracis genotypes are circulating in this region, four strains from various sources isolated from different regions of the Ukraine were investigated. By combining long- and short-read next-generation sequencing techniques, highly accurate genomes were reconstructed, enabling detailed in silico genotyping. Thus, the strains could be assigned to the Tsiankovskii subgroup of the "TransEurAsia" clade, which is commonly found in this region. Their high genetic similarity suggests that the four strains are members of the endemic population whose progenitor was once introduced in the Ukraine and bordering regions. This study provides information on B. anthracis strains from a region where there is little knowledge of the local population, thereby adding to the picture of global B. anthracis genotype distribution. We also emphasize the importance of surveillance and prevention methods regarding anthrax outbreaks, as other studies predicted a higher number of cases in the future due to global warming.

Genomic epidemiology of Campylobacter fetus subsp. venerealis from Germany.

Campylobacter fetus subsp. venerealis (Cfv) causes bovine genital campylobacteriosis (BGC), a World Organization for Animal Health (WOAH)-listed trade-relevant disease characterized by severe reproductive losses, such as infertility, early embryonic death and abortion in cattle. BGC has significant economic implications that have prompted several countries to adopt stringent eradication and surveillance measures to contain the disease. In Germany, there has been a low incidence of BGC cases over the past 28 years. This study aimed to illustrate the genomic diversity of German Cfv strains isolated from different federal states in Germany. This study analyzed 63 Cfv strains, collected between 1985 and 2015, by whole-genome sequencing and compared them with genome data of 91 international Cfv isolates. The phylogenetic analysis showed that the Cfv population is genetically conserved and has geographic clusters. In Germany, one phylogenetic lineage comprising all strains was identified. This German lineage was part of a subclade that probably emerged in the nineteenth century and diversified over time. The results of this study point to a non-recurrent cross-border introduction of Cfv in Germany. The BGC control interventions in Germany can be considered successful as no outbreaks were reported since 2015.

Establishment of a Publicly Available Core Genome Multilocus Sequence Typing Scheme for Clostridium perfringens.

Clostridium perfringens is a spore-forming anaerobic pathogen responsible for a variety of histotoxic and intestinal infections in humans and animals. High-resolution genotyping aiming to identify bacteria at strain level has become increasingly important in modern microbiology to understand pathogen transmission pathways and to tackle infection sources. This study aimed at establishing a publicly available genome-wide multilocus sequence-typing (MLST) scheme for C. perfringens. A total of 1,431 highly conserved core genes (1.34 megabases; 50% of the reference genome genes) were indexed for a core genome-based MLST (cgMLST) scheme for C. perfringens. The scheme was applied to 282 ecologically and geographically diverse genomes, showing that the genotyping results of cgMLST were highly congruent with the core genome-based single-nucleotide-polymorphism typing in terms of resolution and tree topology. In addition, the cgMLST provided a greater discrimination than classical MLST methods for C. perfringens. The usability of the scheme for outbreak analysis was confirmed by reinvestigating published outbreaks of C. perfringens-associated infections in the United States and the United Kingdom. In summary, a publicly available scheme and an allele nomenclature database for genomic typing of C. perfringens have been established and can be used for broad-based and standardized epidemiological studies. IMPORTANCE Global epidemiological surveillance of bacterial pathogens is enhanced by the availability of standard tools and sharing of typing data. The use of whole-genome sequencing has opened the possibility for high-resolution characterization of bacterial strains down to the clonal and subclonal levels. Core genome multilocus sequence typing is a robust system that uses highly conserved core genes for deep genotyping. The method has been successfully and widely used to describe the epidemiology of various bacterial species. Nevertheless, a cgMLST typing scheme for Clostridium perfringens is currently not publicly available. In this study, we (i) developed a cgMLST typing scheme for C. perfringens, (ii) evaluated the performance of the scheme on different sets of C. perfringens genomes from different hosts and geographic regions as well as from different outbreak situations, and, finally, (iii) made this scheme publicly available supported by an allele nomenclature database for global and standard genomic typing.

Epidemiological Analysis of Salmonella enterica subsp. enterica Serovar Dublin in German Cattle Herds Using Whole-Genome Sequencing.

Salmonella enterica subsp. enterica serovar Dublin is a cattle-adapted serovar that causes enteritis and systemic diseases in animals. In Germany, S. Dublin is not detected or is very rarely detected in some federal states but is endemic in certain regions. Information on detailed genetic characteristics of S. Dublin is not available. An understanding of the paths and spreading of S. Dublin within and between regions and over time is essential to establish effective control strategies. Whole-genome sequencing (WGS) and bioinformatic analysis were used to explore the genetic traits of S. Dublin and to determine their epidemiological context. Seventy-four S. Dublin strains collected in 2005 to 2018 from 10 federal states were studied. The phylogeny was analyzed using core-genome single-nucleotide polymorphisms (cgSNPs) and core-genome multilocus sequence typing. Genomic clusters at 100 cgSNPs, 40 cgSNPs, and 15 cgSNPs were selected for molecular epidemiology. WGS-based genoserotyping confirmed serotyping. Important specific virulence determinants were detected in all strains, but multidrug resistance in German S. Dublin organisms is uncommon. Use of different thresholds for cgSNP analysis enabled a broad view and also a detailed view of the occurrence of S. Dublin in Germany. Genomic clusters could be allocated nationwide, to a limited number of federal states, or to special regions only. Results indicate both persistence and spread of S. Dublin within and between federal states in short and longer time periods. However, to detect possible routes of infection or persistence of S. Dublin indicated by genomic analysis, information on the management of the cattle farms and contacts with corresponding farms are essential. IMPORTANCE Salmonella enterica subsp. enterica serovar Dublin is a bovine host-adapted serovar that causes up to 50% of all registered outbreaks of salmonellosis in cattle in Germany. S. Dublin is not detected or is only rarely detected in some federal states but has been endemic in certain regions of the country for a long time. Information on genetic traits of the causative strains is essential to determine routes of infection. WGS and bioinformatic analysis should be used to explore the genetic characteristics of S. Dublin. Combining the genomic features of S. Dublin strains with information on the management of the cattle farms concerned should enable the detection of possible routes of infection or persistence of S. Dublin. This approach is regarded as a prerequisite to developing effective intervention strategies.

Tracking the Distribution of Brucella abortus in Egypt Based on Core Genome SNP Analysis and In Silico MLVA-16.

Brucellosis, caused by the bacteria of the genus Brucella, is one of the most neglected common zoonotic diseases globally with a public health significance and a high economic loss among the livestock industry worldwide. Since little is known about the distribution of B. abortus in Egypt, a total of 46 B. abortus isolates recovered between 2012-2020, plus one animal isolate from 2006, were analyzed by examining the whole core genome single nucleotide polymorphism (cgSNP) in comparison to the in silico multilocus variable number of tandem repeat analysis (MLVA). Both cgSNP analysis and MLVA revealed three clusters and one isolate only was distantly related to the others. One cluster identified a rather widely distributed outbreak strain which is repeatedly occurring for at least 16 years with marginal deviations in cgSNP analysis. The other cluster of isolates represents a rather newly introduced outbreak strain. A separate cluster comprised RB51 vaccine related strains, isolated from aborted material. The comparison with MLVA data sets from public databases reveals one near relative from Argentina to the oldest outbreak strain and a related strain from Spain to a newly introduced outbreak strain in Egypt. The distantly related isolate matches with a strain from Portugal in the MLVA profile. Based on cgSNP analysis the oldest outbreak strain clusters with strains from the UK. Compared to the in silico analysis of MLVA, cgSNP analysis using WGS data provides a much higher resolution of genotypes and, when correlated to the associated epidemiological metadata, cgSNP analysis allows the differentiation of outbreaks by defining different outbreak strains. In this respect, MLVA data are error-prone and can lead to incorrect interpretations of outbreak events.

The fungivorous amoeba Protostelium aurantium targets redox homeostasis and cell wall integrity during intracellular killing of Candida parapsilosis.

Predatory interactions among microbes are major evolutionary driving forces for biodiversity. The fungivorous amoeba Protostelium aurantium has a wide fungal food spectrum including foremost pathogenic members of the genus Candida. Here we show that upon phagocytic ingestion by the amoeba, Candida parapsilosis is confronted with an oxidative burst and undergoes lysis within minutes of processing in acidified phagolysosomes. On the fungal side, a functional genomic approach identified copper and redox homeostasis as primary targets of amoeba predation, with the highly expressed copper exporter gene CRP1 and the peroxiredoxin gene PRX1 contributing to survival when encountered with P. aurantium. The fungicidal activity was largely retained in intracellular vesicles of the amoebae. Following their isolation, the content of these vesicles induced immediate killing and lysis of C. parapsilosis in vitro. Proteomic analysis identified 56 vesicular proteins from P. aurantium. Although completely unknown proteins were dominant, many of them could be categorised as hydrolytic enzymes targeting the fungal cell wall, indicating that fungal cell wall structures are under selection pressure by predatory phagocytes in natural environments. TAKE AWAY: The amoeba Protostelium aurantium feeds on fungi, such as Candida parapsilosis. Ingested yeast cells are exposed to reactive oxygen species. A copper exporter and a peroxiredoxin contribute to fungal defence. Yeast cells undergo intracellular lysis. Lysis occurs via a cocktail of hydrolytic enzymes from intracellular vesicles.